The objectives of the proposed research are to study and elucidate the mechanism of dehydrogenation of fatty acyl CoA. This dehydrogenation requires participation of two separate classes of flavoproteins; a substrate-level dehydrogenase, and another flavoprotein, termed electron-transfer-flavoprotein (ETF). The function of the latter enzyme has always been thought to be limited to electron transfer from acyl CoA-dehydrogenase to the mitochondrial electron transport chain. However, there is evidence ETF participates in the dehydrogenation and that enoyl CoA is not released until ETF is reoxidized. These studies suggest that 2 e to 1 e transduction does not occur between the dehydrogenase and ETF. Attempts will be made to further probe the interaction of these two types of flavoproteins with each other and with substrates, the rest of beta-oxidation enzymes, and with membrane bound components. This will be done by isolating stable and kinetic spectral intermediates and determining the rate and binding constants for each step in the overall process. Techniques such as rapid reaction, anaerobic titration and direct binding as well as absorbance, fluorescence, EPR and Raman spectroscopy will be used to probe the enzyme-mechanism. Studies on a new ETF-linked enzyme from pig liver, isovaleryl CoA dehydrogenase, as well as on a new acyl CoA dehydrogenase from Ps. oleovorans which interacts with pig liver ETF are proposed. Studies on an apparent complex of oxidized short-chain acyl CoA dehydrogenase and an unknown CoA derivative are proposed. These studies will hopefully lead to better understanding of the mechanism and control of this key step in beta-oxidation of fatty acids in mitochondria, and may shed light on the effects of hypoglycemic agents, regulation of beta-oxidation, and interrelationships between beta-oxidation and other major sequences of metabolism such as gluconeogenesis.